Identification of Porcine (Sus scrofa) Sperm Sex Chromosome Gene Expression and Its Biological Function Based on Proteomics
SHI Yong1, LIU Yi-Bing1, LIU Hua-Tao1, WANG Chu-Duan1,*, XING Kai2,*
1 College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; 2 College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China
Abstract：It is critical to the actual output of animal husbandry that the sex control of animals be properly managed. A frequent commodity in the manufacturing of sex-controlled semen is used to regulate the sex ratio of their progeny. The commercial use of sex-controlled semen is mostly concentrated in dairy cows (Bos taurus), and sex-controlled frozen semen plays an important role in the propagation of improved breeds of dairy cows. Both the mechanical damage and the reduction of sperm motility after sorting are inevitable, so immunological methods to sort X/Y sperm is an ideal method. Semen from 10 healthy male boars was collected to study the gene expression of proteins on the sex chromosomes of porcine (Sus scrofa) sperm and its biological function, as well as to search for possible antigens for sex control in porcine sperm. Flow cytometry was used to remove seminal plasma and separate the sperm into X/Y sperm. The protein expression in the isolated porcine sperm was analyzed qualitatively and quantitatively by Label-free proteomics technology, and the expressed genes distributed on the sex chromosome were extracted and analyzed their biological function. Simultaneously, the protein expressed on the sex chromosome was predicted the transmembrane region by TMHMM 2.0. Subcellular localization was predicted by WoLF PSORT, signal peptide was predicted by SignalP 5.0; and some selected antigens were validated. A total of 1 925 proteins and 13 558 peptides were obtained by proteinomic sequencing. According to the results of this study, there were 2 976 proteins-coding genes, of which 67 were expressed by sex-specific genes, with 65 X and 2 Y chromosomes genes. The GO and KEGG analyses of the genes expressed on allosome revealed that they were mostly engaged in the energy generation pathway associated with sperm motility. 45 proteins were expected to contain the transmembrane structure, 47 proteins were located on the plasma membrane or extracellular region, and 28 proteins could generate signal peptides (secretory signal peptides/signal peptides, Sec/SPI). The proteins expressed in deoxyribonuclease like 1 (DNASE1L1), ATPase H+ transporting accessory protein 1 (ATP6AP1), tetraspanin 6 (TSPAN6), NADH: ubiquinone oxidoreductase subunit A1 (NDUFA1), phosphoribosyl pyrophosphate synthetase 2 (PRPS2), NADPH oxidase heavy chain subunit (GP91-PHOX), B cell receptor associated protein 31 (BCA31), sperm acrosome associated 5 (SPACA5), TIMP metallopeptidase inhibitor 1 (TIMP1), peroxiredoxin 4 (PRDX4) and apolipoprotein O like (APOOL) were selected as candidate antigens. The proteins ATP6AP1, NDUFA1, PRPS2, GP91-PHOX and TIMP1 were identified by Western blot. This study provides a reference for studying the genes expressed on the neutral chromosomes of porcine sperm and the strategies of sex control of porcine sperm.
石咏, 刘怡冰, 刘华涛, 王楚端, 邢凯. 基于蛋白组学鉴定猪精子中的性染色体基因表达及其生物学功能[J]. 农业生物技术学报, 2022, 30(9): 1747-1762.
SHI Yong, LIU Yi-Bing, LIU Hua-Tao, WANG Chu-Duan, XING Kai. Identification of Porcine (Sus scrofa) Sperm Sex Chromosome Gene Expression and Its Biological Function Based on Proteomics. 农业生物技术学报, 2022, 30(9): 1747-1762.
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